Transformed cells useful for the control of pests

ABSTRACT

The subject invention provides materials and methods for the control of pests. Specifically exemplified is the use of recombinant hosts to control mosquito larvae. These hosts, which may be, for example yeast or algae, can be transformed so that they express a pesticidal polypeptide which controls mosquito larvae. These transformed microbes can then be applied to surface waters where mosquito larvae are likely to be found.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/295,846, filed on Apr. 21, 1999.

The subject invention was made with government support under researchprojects supported by NIH Grant No. AI 41254-01 and USDA/FAES/FME-03249.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Many blood-ingesting pests are known to feed on humans and animals, andmany pests are vectors for pathogenic microorganisms which threatenhuman and animal health, including commercially important livestock,pets and other animals. Various species of mosquitoes, for example,transmit diseases caused by viruses, and many are vectors fordisease-causing nematodes and protozoa. Mosquitoes of the genusAnopheles transmit Plasmodium, the protozoan which causes malaria, adevastating disease which results in approximately 1 million deathsannually. The mosquito species Aedes aegypti transmits an arbovirus thatcauses yellow fever in humans. Other arboviruses transmitted by Aedesspecies include the causative agents of dengue fever, eastern andwestern encephalitis, Venezuelan equine encephalitis, St. Louisencephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex,which includes the common house mosquito C. pipiens, is implicated inthe transmission of various forms of encephalitis and filarial worms.The common house mosquito also transmits Wuchereria bancrofti and Brugiamalayi, which cause various forms of lymphatic filariasis, includingelephantiasis. Trypanasoma cruzi, the causative agent of Chagas'disease, is transmitted by various species of blood-ingestingTriatominae bugs. The tsetse fly (Glossina spp.) transmits Africantrypanosomal diseases of humans and cattle. Many other diseases aretransmitted by various blood-ingesting pest species. The order Dipteracontains a large number of blood-ingesting and disease-bearing pests,including, for example, mosquitoes, black flies, no-see-ums (punkies),horse flies, deer flies and tsetse flies.

Various pesticides have been employed in efforts to control or eradicatepopulations of disease-bearing pests, such as disease-bearingblood-ingesting pests. For example, DDT, a chlorinated hydrocarbon, hasbeen used in attempts to eradicate malaria-bearing mosquitoes throughoutthe world. Other examples of chlorinated hydrocarbons are BHC, lindane,chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin,dieldrin, chlordane, heptachlor, and endrin). The long-term stability ofmany of these pesticides and their tendency to bioaccumulate render themparticularly dangerous to many non-pest organisms.

Another common class of pesticides is the organophosphates, which isperhaps the largest and most versatile class of pesticides.Organophosphates include, for example, parathion, Malathion™, diazinon,naled, methyl parathion, and dichlorvos. Organophosphates are generallymuch more toxic than the chlorinated hydrocarbons. Their pesticidaleffect results from their ability to inhibit the enzyme cholinesterase,an essential enzyme in the functioning of the insect nervous system.However, they also have toxic effects on many animals, including humans.

The carbamates, a relatively new group of pesticides, include suchcompounds as carbamyl, methomyl, and carbofuran. These compounds arerapidly detoxified and eliminated from animal tissues. Their toxicity isthought to involve a mechanism similar to the mechanism of theorganophosphates; consequently, they exhibit similar shortcomings,including animal toxicity.

A major problem in pest control results from the capability of manyspecies to develop pesticide resistance. Resistance results from theselection of naturally-occurring mutants possessing biochemical,physiological or behavioristic factors that enable the pests to toleratethe pesticide. Species of Anopheles mosquitoes, for example, have beenknown to develop resistance to DDT and dieldrin. DDT substitutes, suchas Malathion™, propoxur and fenitrothion are available; however, thecost of these substitutes is much greater than the cost of DDT.

There is clearly a longstanding need in the art for pesticidal compoundsthat are pest- specific, that reduce or eliminate direct and/or indirectthreats to human health posed by presently available pesticides, thatare environmentally compatible in the sense that they are biodegradable,and are not toxic to non-pest organisms, and have reduced or no tendencyto bioaccummulate.

Many pests, including for example blood-inbibing pests, must consume anddigest a proteinaceous meal to acquire sufficient essential amino acidsfor growth, development and the production of mature eggs. Adult pests,such as adult mosquitoes, need these essential amino acids for theproduction of vitellogenins by the fat body. These vitellogenins areprecursors to yolk proteins which are critical components of oogenesis.Many pests, such as house flies and mosquitoes, produce oostatichormones that inhibit egg development by inhibiting digestion of theprotein meal, and thereby limiting the availability of the essentialamino acids necessary for egg development.

Serine esterases such as trypsin and trypsin-like enzymes (collectivelyreferred to herein as “TTLE”) are important components of the digestionof proteins by insects. In the mosquito, Aedes aegypti, an early trypsinthat is found in the midgut of newly emerged females is replaced,following the blood meal, by a late trypsin. A female mosquito typicallyweighs about 2 mg and produces 4 to 6 μg of trypsin within several hoursafter a ingesting blood meal. Continuous boisynthesis at this rate wouldexhaust the available metabolic energy of a female mosquito; as aresult, the mosquito would be unable to produce mature eggs, or even tofind an oviposition site. To conserve metabolic energy, the mosquitoregulates TTLE biosynthesis with a peptide hormone named TrypsinModulating Oostatic Factor (TMOF). Mosquitoes produce TMOF in thefollicular epithelium of the ovary 12-35 hours after a blood meal; TMOFis then released into the hemolymph where it binds to a specificreceptor on the midgut epithelial cells, signaling the termination ofTTLE biosynthesis.

This regulatory mechanism is not unique for mosquitoes; flesh flies,fleas, sand flies, house flies, dog flies and other pests which ingestprotein as part of their diet have similar regulatory mechanisms.

In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosedthat injection of a hormone preparation into the body cavity of bloodimbibed mosquitoes caused inhibition of egg development and sterility(Borovsky, D. [1985] Arch. Insect Biochem. Physiol. 2:333-349).Following these observations, Borovsky (Borovsky, D. [1988] Arch. Ins.Biochem. Physiol. 7:187-210) reported that injection or passage of apeptide hormone preparation into mosquitoes inhibited the TTLEbiosynthesis in the epithelial cells of the gut. This inhibition causedinefficient digestion of the blood meal and a reduction in theavailability of essential amino acids translocated by the hemolymph,resulting in arrested egg development in the treated insect. Borovskyobserved that this inhibition of egg development does not occur when theoostatic hormone peptides are inside the lumen of the gut or other partsof the digestive system (Borovsky, D. [1988], supra).

Following the 1985 report, the isolated hormone, (a ten amino acidpeptide) and two TMOF analogues were disclosed in U.S. Pat. Nos.5,011,909 and 5,130,253, and in a 1990 publication (Borovsky, et al.[1990] FASEBJ 4:3015-3020). Additionally, U.S. Pat. No. 5,358,934discloses truncated forms of the full length TMOF which have prolinesremoved from the carboxy terminus, including the peptides YDPAP (SEQ IDNO. 14), YDPAPP (SEQ ID NO. 15), YDPAPPP (SEQ ID NO. 16), and YDPAPPPP(SEQ ID NO. 17).

Neuropeptides Y (NPY) are an abundant family of peptides that are widelydistributed in the central nervous system of vertebrates. NPY peptideshave also recently been isolated and identified in a cestode, aturbellarian, and in terrestrial and marine molluscs (Maule et al., 1991“Neuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide fromMoniezia expansa (Cestoda: Cyclophylidea)” Parasitology 102:309-316;Curry et al., 1992 “Neuropeptide F: Primary Structure from theTurbellarian, Arthioposthia triangulata” Comp. Biochem. Physiol.101C:269-274; Leung et al., 1992 “The Primary Structure of NeuropeptideF (NPF) from the Garden Snail, Helix aspersa” Regul. Pep. 41:71-81;Rajpara et al., 1992 “Identification and Molecular Cloning ofNeuropeptide Y Homolog that Produces Prolonged Inhibition in AplysiaNeurons” Neuron. 9:505-513).

Invertebrate NPYs are highly homologous to vertebrate NPYs. The majordifference between vertebrate and invertebrate NPYs occurs at theC-terminus where the vertebrate NPY has an amidated tyrosine (Y) whereasinvertebrates have an amidated phenylalanine (F). Because of thisdifference, the invertebrate peptides are referred to as NPF peptides.

Cytoimmunochemical analyses of NPY peptides suggest that they areconcentrated in the brain of various insects, including the Coloradopotato beetle Leptinotarsa decemlineata (Verhaert et al., 1985 “DistinctLocalization of FMRFamide- and Bovine Pancreatic Polypeptide-LikeMaterial in the Brain, Retrocerebal Complex and Subesophageal Ganglionof the Cockroach Periplaneta americana” L. Brain Res. 348:331-338;Veenstra et al., 1985 “Immunocytochemical Localization of PeptidergicNeurons and Neurosecretory Cells in the Neuro-Endocrine System of theColorado Potato Beetle with Antisera to Vertebrate Regulatory Peptides”Histochemistry 82:9-18). Partial purification of NPY peptides in insectssuggests that both NPY and NPF are synthesized in insects (Duve et al.,1981 “Isolation and Partial Characterization of PancreaticPolypeptide-like Material in the Brain of the Blowfly alliphoravomitoria” Biochem. J. 197, 767-770).

Researchers have recently isolated two neuropeptides with NPF-likeimmunoreactivity from brain extracts of the Colorado potato beetle. Theresearchers purified the peptides using C₁₈ reversed phase high pressureliquid chromatography (HPLC), and determined their structure using massspectrometry. The deduced structures of these peptides are:Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) designated NPF Iand NPF II, respectively (Spittaels, Kurt et al. [1996] “InsectNeuropeptide F (NPF)-Related Peptides: Isolation from Colorado PotatoBeetle (Leptinotarsa decemlineata) Brain,” Insect Biochem. Molec. Biol.26(4):375-382).

BRIEF SUMMARY OF THE INVENTION

The subject invention provides materials and methods useful for thecontrol of pests. In a specific embodiment the methods of the subjectinvention can be used for treating mosquito larvae to control mosquitopopulations. Specifically exemplified are recombinant hosts transformedto comprise and/or produce biological control agents capable ofincreasing the mortality of pests, including mosquitoes and mosquitolarvae.

One aspect of the subject invention pertains to a composition comprisinga host edible by mosquito larvae, wherein the cells of the host comprisea biological control agent that increases the mortality of the mosquitolarvae. In a specific embodiment, the biological control agent inhibitsbiosynthesis of digestive enzymes, such as TTLE, thereby inhibiting fooddigestion. This inhibition of food digestion ultimately results instarvation and death of the mosquito larvae.

In a preferred aspect of the subject invention, an appropriate host istransformed with a polynucleotide encoding a polypeptide which acts toinhibit TTLE biosynthesis. Appropriate hosts include, but are notlimited to, prokaryotic and eukaryotic cells, edible by pests includingmosquito larvae. The biological control agents useful according to thesubject invention include, but are not limited to, TMOF or functionalequivalents thereof, NPF or functional equivalents thereof, and otheragents identifiable by, for example, assays employing a TMOF receptor.

In a specific embodiment of the subject invention, salt water Chlorella,such as Chlorella desiccata are stably transformed with a plasmidcarrying DNA encoding TMOF.

One embodiment of the present invention concerns a pesticide compositioncomprising a peptide having the formula:

A¹A²A³A⁴A⁵Fl (Formula I) (SEQ ID NO. 8)

wherein:

A¹ is selected from the group consisting of A, D, F, G, M, P, S and Y;

A² is selected from the group consisting of A, D, E, F, G, N, P, S andY;

A³ is selected from the group consisting of A, D, F, G, L, P, S and Y;

A⁴ is optionally present when A³ is present and is selected from thegroup consisting of A, F, G, L and Y;

A⁵ is optionally present when A⁴ is present and is selected from thegroup consisting of A, F, L and P;

Fl is a flanking region which is optionally present and is selected fromthe group consisting of: P, PP, PPP, PPPP (SEQ ID NO. 9), and PPPPP (SEQID NO. 10).

Preferably, the peptide does not comprise YDPAP₆ (SEQ ID NO. 11), DYPAP₆(SEQ ID NO. 12), PAP₆ (SEQ ID NO. 13), YDPAP (SEQ ID NO. 14), YDPAP₂(SEQ ID NO. 15), YDPAP₃ (SEQ ID NO. 16), YDPAP₄ (SEQ ID NO. 17), NPTNLH(SEQ ID NO. 18) or DF-OMe.

In a more specific aspect the peptide or protein comprises an amino acidsequence which consists essentially of the amino acid sequence ofFormula 1. In a preferred aspect, the amino acid sequence is a TMOF orNPF fragment and lacks TMOF or NPF amino acids adjacent to the aminoacid sequence of Formula I. Preferably the fragment has from 2-5 aminoacids of TMOF. In still another aspect, the peptide consists of theamino acid sequence of Formula I.

In various embodiments, either A³A⁴A⁵, A³A⁴A⁵Fl, A⁴A⁵,Fl, A⁵ or A⁵Fl arenot present. Where A⁵ is not present, Fl may be attached directly to A⁴.Where A⁴A⁵ is not present, Fl may be attached directly to A³. Finally,where A³A⁴A⁵ is not present, Fl may be attached directly to A².

Preferred peptides are selected from the group consisting of: AAP (SEQID NO. 19), ADP (SEQ ID NO. 20), ADPAP (SEQ ID NO. 21), APA (SEQ ID NO.22), DAA (SEQ ID NO. 23), DF (SEQ ID NO. 24), DPA (SEQ ID NO. 25), DY(SEQ ID NO. 26), DYP (SEQ ID NO. 27), FAP (SEQ ID NO. 28), FDP (SEQ IDNO. 29), FDPAP (SEQ ID NO. 30), FSP (SEQ ID NO. 31), MPDYP5 (SEQ ID NO.32), PAA (SEQ ID NO. 33), PAP (SEQ ID NO. 34), Y(D)DP (SEQ ID NO. 35),Y(D)DPAP (SEQ ID NO. 36), YAP (SEQ ID NO. 37), YD (SEQ ID NO. 38), YDA(SEQ ID NO. 39), YDAAP (SEQ ID NO. 40), YDF (SEQ ID NO. 41), YDFAP (SEQID NO. 42), YDG (SEQ ID NO. 43), YDLAP (SEQ ID NO. 44), YDP (SEQ ID NO.45), (D)YDP (SEQ ID NO. 46), YDPAF (SEQ ID NO. 47), YDPAL (SEQ ID NO.48), (D)YDPAP (SEQ ID NO. 49), YDPFP (SEQ ID NO. 50), YDPGP (SEQ ID NO.51), YDPLP (SEQ ID NO. 52), YEPAP (SEQ ID NO. 53), YFPAP (SEQ ID NO.54), YNPAP (SEQ ID NO. 55) and YSF (SEQ ID NO. 56).

A further embodiment of the present invention comprises a peptide havingthe formula

A¹A² (Formula II) (SEQ ID NO. 62)

wherein

A¹ is an amino acid selected from the group consisting of A, D, F, M,and Y, and

A² is an amino acid selected from the group consisting of A, D, E, P,and Y.

In a preferred embodiment, the subject invention is directed to peptidesof Formula II wherein A¹ and A² are independently selected from thegroup consisting of A, D, and Y.

Specifically exemplified as another embodiment are methods using an NPFpeptide having the sequenceAla-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) orAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2).

The term “pesticidal polypeptide” is used herein to indicate NPF andTMOF peptides, as well as fragments, derivatives and analogues andfunctional equivalents of NPF and TMOF. The present invention alsoprovides analogues of the pesticidal polypeptides which have one or moreamino acid substitutions forming a branched peptide (e.g., bysubstitution with an amino acid or amino acid analogue having a freeamino- or carboxy-side chain that forms a peptide bond with a sequenceof one or more amino acids, including but not limited to prolines) orallowing circularization of the peptide (e.g., substitution with acysteine, or insertion of a cysteine at the amino- or carboxy-terminusor internally, to provide a sulfhydryl group for disulfide bondformation).

The pesticidal polypeptides of the present invention are particularlyadvantageous because their smaller size permits more rapid and efficientpenetration into the midgut. In addition, they are less expensive toproduce by conventional chemical methods.

In one embodiment, the subject invention provides pesticidalpolypeptides having a C-terminus arginine. In a preferred embodiment,these short polypeptides can be joined to form polymers of repeatingunits. Specifically exemplified herein is the (DPAR)₄ (SEQ ID NO. 61)polymer which can be broken into four DPAR (SEQ ID NO. 60) units in thegut of the pest. Advantageously, the short pesticidal polypeptidesconnected by arginine (or other readily cleavable residue) can penetratethe midgut of the pest efficiently.

Also included in this invention are addition salts, complexes, orprodrugs such as esters of the pesticidal polypeptide, especially thenontoxic pharmaceutically or agriculturally acceptable acid additionsalts. The acid addition salts can be prepared in standard manner in asuitable solvent from the parent compound and an excess of an acid, suchas hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic,succinic, ethanedisulfonic or methanesulfonic acids. Also, theN-terminus and C-terminus of the pesticidal polypeptides can bechemically modified to further inhibit proteolysis by metabolic enzymes.

The analogues of the pesticidal polypeptides of the present inventionalso include polypeptides having NPF and/or TMOF amino acid sequences inwhich one or more of the amino acid residues has been substituted by anamino acid in the D- conformation. The presence of D-conformation aminoacids can inhibit the ability of proteases to degrade the peptides ofthe subject invention. Polypeptides having the above sequences in whichonly conservative substitutions have been made are also provided by thepresent invention.

Also, derivation of the pesticidal polypeptides with long chainhydrocarbons will facilitate passage through the cuticle into the pestbody cavity. Accordingly, a further embodiment of the subject inventionpertains to compositions comprising the pesticidal polypeptides bound tolipids or other carriers.

Yet another aspect of the subject invention pertains to polynucleotidesencoding the pesticidal polypeptides of the subject invention. Thesepolynucleotides can readily be synthesized by a person skilled in theart. The sequences may be used to transform an appropriate host toconfer upon that host the ability to express the novel peptides. Hostsof particular interest include bacteria, algae, yeasts, and plants.Viruses may also be modified to comprise polynucleotide sequencesencoding the pesticidal polypeptides of the present invention. For eachof these hosts, the polynucleotides may be specifically designed by aperson skilled in the art to utilize codons known to be optimallyexpressed in the particular hosts. Advantageous promoters are alsoreadily incorporated into the polynucleotides. Bacteria, yeasts, plants,algae, viruses, and other hosts each may be used to produce peptides forfurther use, or these hosts can be used as vehicles for directapplication of the peptide to the target pest. Plants can be transformedto make the plant toxic to a target pest species which feeds on thatplant. Methods for transforming plant cells utilizing, for example,Agrobacteria are well known to those skilled in the art.

As used herein, the term “pesticidally effective” is used to indicate anamount or concentration of a pesticide which is sufficient to reduce thenumber of pests in a geographical area, as compared to a correspondinggeographical area in the absence of the amount or concentration of thepesticide.

The term “pesticidal” is not intended to refer only to the ability tokill pests, but also includes the ability to interfere with a pest'slife cycle in any way that results in an overall reduction in the pestpopulation. For example, the term “pesticidal” included inhibition orelimination of reproductive ability of a pest, as well as inhibition ofa pest from progressing from one form to a more mature form, e.g.,transition between various larval instars or transition from larvae topupa or pupa to adult. Further, the term “pesticidal” is intended toinclude all phases of a pest life cycle; thus, for example, the termincludes larvicidal, ovicidal, and adulticidal action.

The word “transform” is broadly used herein to refer to introduction ofan exogenous polynucleotide sequence into a prokaryotic or eukaryoticcell by any means known in the art (including, for example, directtransmission of a polynucleotide sequence from a cell or virus particleas well as transmission by infective virus particles and transmission byany other known means for introducing a polynucleotide into a cell),resulting in a permanent or temporary alteration of genotype and in animmortal or non-immortal cell line.

The terms “peptide,” “polypeptide,” and “protein” as used herein areintended to refer to amino acid sequences of any length.

Another aspect of the subject invention pertains to a method ofcontrolling pests comprising administering to said pest an effectiveamount of a peptide of the subject invention.

The subject invention provides pesticidal compositions wherein thepesticidal polypeptides are formulated for application to the targetpests, or their situs. In a specific embodiment, the present inventionprovides recombinant hosts which express a polynucleotide encoding apesticidal polypeptide to produce the pesticidal polypeptide. Therecombinant host may be, for example, prokaryotic or eukaryotic. In aspecific example, yeast or algae are transformed to express a pesticidalpolypeptide of the subject invention. The transformed hosts are thenapplied to water areas where mosquito larvae will ingest the transformedhost resulting in control of the mosquitoes by the pest control agent.

Preferably, the subject peptides have an LD₅₀ against mosquito larvae ofless than 3.0 μmoles/ml. More preferably, the peptides have an LD₅₀ ofless than 2.0 μmoles/ml, and, most preferably, the peptides have an LD₅₀of less than 1.0 μmoles/ml. As used herein, “LD₅₀” refers to a lethaldose of a peptide able to cause 50% mortality of larvae maintained on adiet of 1 mg/ml autoclaved yeast (Borovsky and Mahmood [1995] “Feedingthe mosquito Aedes aegypti with TMOF and its analogs; effect on trypsinbiosynthesis and egg development,” Regulatory Peptides 57:273-281).

Another aspect of the subject invention pertains to methods ofcontrolling pests comprising preparing a host to produce a pesticidalpolypeptide wherein the host is edible by the target pest andadministering the host to the target pest.

Another aspect of the subject invention pertains to pesticidalpolypeptides and other pesticidal compounds used in conjunction with amarker to aid in the administration and/or monitoring of the biologicalcontrol agent. Such markers include, for example, Green FluorescentProtein (GFP). This protein fluoresces and provides a means to determinewhether target pests, such as mosquitoes, are eating or have beentreated by the biological control agent. Target pests which have eatenthe biological control agent fused with the GFP will fluoresce. Thisfluorescence can be employed as an analytical measurement whichindicates whether target pests such as mosquito larvae are indeedconsuming the pesticidal polypeptide or other pesticidal compound Suchmeasurements are useful for determining the amount of pesticidalpolypeptide which must be applied to maintain a pesticidally effectiveamount of the pesticidal polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the construction of the pYDB2 plasmid.

FIGS. 2A and 2B depict the introduction of the pYDB2 plasmid into yeast.

FIG. 3 shows the induction of TMOF expression (in the transformed yeast)using ELISA.

FIG. 4 shows the inhibition of A. aegypti larvae by ingestion oftransformed yeast expressing the TMOF-GFP fusion protein. Survival ofthe larvae dropped to less than 20% after 144 hours. Controls exhibitedno decrease in survival.

FIG. 5 shows the inhibition of A. aegypti larvae by ingestion of heatinactivated transformed yeast containing the TMOF-GFP gene. Survivaldropped to less than 20% after 144 hours. Controls show no decrease insurvival.

FIG. 6 depicts the pKylx71 plasmid containing the TMOF-GFP gene.

FIG. 7 shows the inhibition of A. aegypti larvae after ingestion ofChlorella transformed with the GFP-TMOF gene. Survival of the larvaedropped to less than 10% after 72 hours. Controls showed no significantdecrease in survival rate.

FIG. 8 shows a structure of plasmid pYES2/TMOF, including the 2 μ oridirection of replication from the 35S² promoter, TMOF gene cloning siteand the direction of replication of the Neomycin gene.

FIG. 9 shows a structure of pYDB3 including the direction of replicationof TMOF from the 35S² promoter, the cloning site of TMOF and the 2.3 Kbnitrate reductase gene (NR1) and the direction of replication of theNeomycin gene.

FIG. 10 shows a structure of pYDB4 including the direction ofreplication of TMOF gene from the 35S² promoter and the direction ofreplication of the Neomycin resistant gene and the position of the 8.0Kb nitrate reductase gene.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is a neuropeptide designated NPF I.

SEQ ID NO. 2 is a neuropeptide designated NPF II.

SEQ ID NO. 3 is a polynucleotide encoding an amino acid sequence of aTMOF receptor.

SEQ ID NO. 4 is an amino acid sequence of a TMOF receptor.

SEQ ID NO. 5 is a forward primer useful according to the subjectinvention.

SEQ ID NO. 6 is a backward primer useful according to the subjectinvention.

SEQ ID NO. 7 is an oligonucleotide useful according to the subjectinvention.

SEQ ID NO. 8-59 are TMOF peptides useful according to the subjectinvention.

SEQ ID NO. 60-61 are TMOF-R analogue peptides useful according to thesubject invention.

SEQ ID NO. 62 is a TMOF peptide useful according to the subjectinvention.

SEQ ID NO. 63 is an unamidated version of the neuropeptide designatedNPF I (SEQ ID NO. 1).

SEQ ID NO. 64 is an unamidated version of the neuropeptide designatedNPF II (SEQ ID NO. 2).

SEQ ID NO. 65 is the TMOF synthetic gene sense strand useful accordingto the subject invention.

SEQ ID NO. 66 is the TMOF synthetic gene antisense strand usefulaccording to the subject invention.

SEQ ID NO. 67-71 are TMOF-R analogue peptides useful according to thesubject invention.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention is directed to materials and methods forcontrolling pest populations. Specifically exemplified herein arecompositions comprising hosts which contain, are associated with, and/orwhich express pesticidal polypeptides and other biological controlagents. Also exemplified herein are methods of controlling pests, suchas mosquitoes, employing hosts which contain, are associated with,and/or which express the pesticidal polypeptides or other pesticidalcompounds. Preferably, the pesticidal agents have the ability to inhibitbiosynthesis of TTLE. The term “pest” as used herein includesmosquitoes, insects and other organisms which adversely affect humans,plants or animals. This includes pests that remove blood, tissue or anyother fluid from their prey or host. Pests controlled according to thesubject invention include those which have a mechanism for regulatingdigestive enzymes, such as TTLE, which mechanism involves the binding ofa ligand to a receptor to increase or decrease such enzymes, e.g., TMOFor NPF binding to its receptor. Examples of pests which can becontrolled according to the subject invention include, but are notlimited to, mosquitoes, fleshflies, fleas, sandflies, houseflies,dogflies, and insects which attack plants.

In a preferred embodiment, the present invention provides a host cell orvirus particle which contains, is associated with and/or produces apesticidal polypeptide (a “pesticidal cell”). The host can be aprokaryotic or eukaryotic cell which is transformed to express apesticidal polypeptide or other biological control agent. The host mayalso be a viral particle which has been prepared to deliver apolynucleotide encoding a pesticidal polypeptide to a cell. The host canbe prepared by transforming the host with a polynucleotide encoding apeptide capable of inhibiting biosynthesis of digestive enzymes, such asTTLE. In specific embodiments, the host cell is Chlorella or yeast andthe peptide is TMOF, NPF, a TMOF receptor-binding compound, or afunctional analogue, derivative, fragment or other functional equivalentof the TMOF, NPF or other TMOF receptor-binding compound.

The pest control compositions according to the subject invention includecompounds which comprise an NPF or TMOF peptide or a TMOFreceptor-binding compound, or analogue, derivative, fragment or otherfunctional equivalent of the TMOF, NPF or other TMOF receptor-bindingcompound (collectively referred to herein as “pesticidal polypeptides”).One or more pesticidal polypeptides may be provided as a component of aformulation, or as the sole component of the pesticidal composition. Thepesticidal compositions may further comprise a carrier solution,compound, or molecule. Pesticidal compositions of the subject inventionmay also comprise pesticidal polypeptides contained in or associatedwith a prokaryotic or eukaryotic cell, such as a plant, animal or fungicell, and may also be contained in or associated with a viral particle.Examples include, but are not limited to, transformed bacteria, animalcells, algae, fungi, yeast, viruses, and plants that comprise apolynucleotide encoding a pesticidal polypeptide.

The term “functional equivalent” as used herein refers to a full-lengthNPF or TMOF peptide, or an analogue, derivative, fragment or extensionthereof which retains some or all of the biological activity of thecorresponding NPF or TMOF peptide. Functional equivalents also include,for example, an NPF or TMOF peptide in a salt, complex, analogue, orderivative form. The term “NPF polypeptide” refers to compounds whichcomprise an NPF peptide and includes analogues, fragments, derivativesand other functional equivalents thereof. The term “TMOF polypeptide”refers to compounds which comprise a TMOF peptide and includesanalogues, fragments, derivatives and other functional equivalentsthereof.

The pesticidal polypeptides of the subject invention may be presented asfusion proteins or peptides, the amino acid sequence of which includesone or more polypeptides of the present invention and may optionallyinclude one or more heterologous polypeptides. In various specificembodiments, two or more of the polypeptides are linked, for example, bypeptide bonds between the N-terminus of one portion and the C-terminusof another portion. In other aspects, one or more of the polypeptidescan be linked to one or more heterologous peptides or proteins to formpesticidal fusion peptides. Molecules comprising such portions linked byhydrocarbon linkages are also provided. Derivatives of the foregoingfusion proteins are also provided (e.g., branched, cyclized, N- orC-terminal chemically modified, etc.).

Pesticidal polypeptides in which only conservative substitutions havebeen made are also provided by the present invention. The presentinvention also provides analogues which have one or more amino acidsubstitutions forming a branched peptide (e.g., by substitution with anamino acid or amino acid analogue having a free amino- or carboxy-sidechain that forms a peptide bond with a sequence of one or more aminoacids including, but not limited to, prolines) or allowingcircularization of the peptide (e.g., by substitution with a cysteine orinsertion of a cysteine at the amino- or carboxy-terminus or internallyto provide a sulfhydryl group for disulfide bond formation), are alsoprovided.

Nonclassical amino acids or chemical amino acid analogues can beintroduced as a substitution, insertion or addition into the pesticidalpolypeptides of the present invention. Non-classical amino acids includebut are not limited to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, C-methyl amino acids, N-methyl aminoacids, and amino acid analogues in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary). Dextrorotary amino acids areindicated herein by a parenthetical D, i.e., “(D)”, immediatelypreceding the dextrorotary amino acid.

Thus, the pesticidal polypeptides, analogues, fragments, derivatives,and other functional equivalents thereof, include peptides containing,as a primary amino acid sequence, all or part of an exemplifiedpolypeptide sequence including altered sequences in which functionallyequivalent amino acid residues are substituted for residues within thesequence resulting in a peptide which is functionally active. Forexample, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity which acts as afunctional equivalent, resulting in a silent alteration. Conservativesubstitutions for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs (see Table1). The pesticidal polypeptides, and fragments, derivatives andanalogues thereof can be made by chemical peptide synthesis or byrecombinant production from a polynucleotide encoding the pesticidalpolypeptides.

In a specific embodiment, the subject invention is directed toward amethod of controlling blood-ingesting pests comprising preparing atreatment comprising NPF and/or TMOF compounds and applying saidtreatment to said blood-ingesting pests. In another embodiment thesepeptides are used to control agricultural pests.

The transformed pesticidal polypeptide-producing host can beadministered by bringing the host into contact with the habitat of atarget pest. For example, where the target pest is a mosquito, thepesticidal polypeptide can be applied to water in environments known tobe natural habitats for mosquitoes and mosquito larvae. In oneembodiment, the host is alive and will proliferate in the environment.Alternatively, the host may be applied in a non- living state.Preferably, the host is palatable to the target pest leading to theingestion of the transformed host. Once in the gut of the target, thehost may further divide and grow, but many host cells will lyse. Thislysis releases the biological control agent in the gut of the targetpest, ultimately resulting in the death of the pest.

Mosquitoes are preferred target pests according to the presentinvention. Mosquito larvae grow in wet environments, such as marshes orponds, and they consume cells, typically found in such environments.Algae typically comprise a significant portion of a mosquito larvae'sdiet, and as a result, algae are preferred organisms which can betransformed according to the subject invention to express agents knownto inhibit biosynthesis of TTLE. Thus, in one embodiment, the presentinvention is directed to a composition comprising an algae celltransformed to express a pesticidal polypeptide. The algae cell ispreferably a unicellular algae and is most preferably a unicellulargreen algae. With the benefit of the teachings provided herein, algae,such as Chlorella, can be transformed to express pesticidalpolypeptides. In a specific embodiment, the microbe that is transformedis a salt water Chlorella such as Chlorella desiccata.

Preferably, the algae is transformed with a polynucleotide encoding apesticidal polypeptide having the ability to inhibit biosynthesis of oneor more pest digestive enzymes such as TTLE. As used herein, referenceto “functional equivalents” includes, without limitation, truncated orextended TMOF or NPF polypeptides (as compared to native NPF or TMOFpolypeptides) as well as analogues and derivatives of NPF and TMOFpolypeptides, where such truncated peptides, extended peptides,analogues and derivatives exhibit partial, equal or improved functionalcharacteristics as compared to the native NPF and TMOF polypeptides.Additionally, the pesticidal polypeptides of the present inventioninclude non-TMOF and non-NPF polypeptides and other compounds which bindto a TMOF or NPF receptor. Thus, the peptide can be a protein or othermolecule as identified through, for example, assays employing a TMOFreceptor described herein.

Further, to aid in the administration and quantification of delivery ofthe transformed host, various markers can be used to test whether thetransformed host exists in vitro or in the environment and/or whetherpests, such as mosquito larvae, have ingested the pesticidal host. Oneexample of such a marker is the Green Fluorescent Protein (GFP). Usingknown techniques, a fusion protein containing both the GFP and abiological control agent of the subject invention can be prepared. Uponingestion of a host containing this fusion protein, the mosquito larvaewill fluoresce. This fluorescence facilitates quantification of theamount of host administered and the amount of host being eaten by themosquito larvae, which, permits adjustment of the application rate ofthe transformed hosts to ensure the continued presence of a pesticidallyeffective concentration of the host in the environment of the targetpest. The amount of host administered can thereby be precisely refined,improving the efficiency of product use and reducing overall costs oftreatment.

In an alternative embodiment, the host can be a synthetic capsulecontaining a biological control agent. The capsule can comprise, forexample, a microcontainer such as a microsphere having an outer shellwhich can degrade when exposed to the conditions of the larvae gut.Materials suitable for selective degrading under such conditions arenumerous and commonly known in the art. These microcontainers can bedeposited in larvae habitats whereby they will be passively ingestedduring the course of normal feeding.

In addition, there are numerous agents known to infect insects such asmosquitoes. These agents can, for example, comprise certain types ofbacteria and viruses, such as baculoviruses and entomopoxviruses. Uponinfection of a pest organism, the production of biological controlagents by the pest's own cells will result in the death of the pest.

Various biological control agents can be used in the compositions of thesubject invention. For example, U.S. Pat. Nos. 5,011,909; 5,130,253; and5,358,934 describe polynucleotides encoding TMOF and functionalequivalents thereof.

In one embodiment, the subject invention pertains to the pesticidal useof peptides which comprise the amino acid sequence of Formula I (SEQ IDNO. 8). In a preferred embodiment, the subject invention is directed topeptides which comprise the amino acids A, D, and Y. Preferably, thesubject peptides have an LD₅₀ against mosquito larvae of less than 3.0μmoles/ml. More preferably, the peptides have an LD₅₀ of less than 2.0μmoles/ml, and, most preferably, the peptides have an LD₅₀ of less than1.0 μmoles/ml. As used herein, “LD₅₀” refers to a lethal dose of apeptide able to cause 50% mortality of larvae maintained on a diet of 1mg/ml autoclaved yeast (Borovsky and Mahmood [1995] supra).

In one embodiment, the pesticidal polypeptide comprises a repeating unitof at least 3 amino acids. There may be, for example, from 2 to 10 ormore repeating units. Preferably, the repeating unit is connectedthrough at least one amino acid which is cleaved by a pest gut enzyme.As used herein, a pest gut enzyme is an enzyme which is present in thegut of a pest. Preferably, the pest is a mosquito or a lepidopteran. Ina specific embodiment the repeating units are connected through anarginine.

TMOF analogues (5 to 8 amino acids in length, and polymers of theseanalogues) in which Arg was added at the C-terminus were evaluated todetermine their effect on larval growth and development. A series ofanalogues were synthesized and tested by feeding them to mosquito larvaeat concentrations of (0.5 to 5.0 mg/ml; Table 4). Concentrations of 2.0to 0.065 mg/ml were used to feed mosquito larvae and calculate theLethal Dose at 50% mortality (LD₅₀; Table 4) of the TMOF analogues.Several analogues that were effective at LC₅₀ of 0.24 to 0.048 mM werechosen and were injected into 2^(nd) instar Heliothis virescens and theinhibition of trypsin biosynthesis was followed for 24 hours (Table 5).In both cases larval death and trypsin biosynthesis inhibition was noted(Table 4 and 5). These results indicate that short TMOF analogues orpolymers of these analogues with Arg at the termini can be usedefficiently to block larval growth by shutting down the enzyme thatdigests the food in both mosquitoes and Heliothis. The advantage ofusing short analogues connected by Arg is that they can be digested inthe gut into short TMOF analogues that can penetrate the midgut muchfaster than longer analogues.

The skilled artisan, having the benefit of the instant description, canuse techniques well-known in the art to transform a host to express thepesticidal polypeptides of the present invention. The transformed hostthen expresses the polynucleotides.

In a specific embodiment exemplified herein, yeast cells that weretransformed to express GFP-TMOF were fed to mosquito larvae and comparedwith controls in which the GFP-TMOF gene was not part of the yeastgenome. Larvae fed on either (1) live recombinant yeast cells, or (2)heat inactivated cells, starved and died within 5 to 6 days. Incontrast, controls fed on normal yeast cells grew and developednormally, producing adult mosquitoes.

These results show that recombinant yeast can be utilized as a vehicleto control mosquito populations. Using yeast cells that synthesize GFPhas the advantage that the amount of pesticidal polypeptide consumed bymosquito larvae can be determined by measuring the fluorescence of themosquito larvae. This information permits users to ensure that thepesticidal compositions are applied at a sufficient rate to adequatelycontrol the mosquito population.

TMOF Receptors and Polynucleotides

In one embodiment, the subject invention is directed to the control ofpests using a pesticidal polypeptide or other compound which binds to orotherwise associates with a TMOF receptor. Specifically exemplifiedherein is a TMOF receptor comprising the amino acid sequence shown inSEQ ID NO. 4. Preferably, the polypeptide is encoded by a completenucleotide sequence of a TMOF receptor gene or fragments or mutantsthereof which encode polypeptides having TMOF receptor activity. In aspecific embodiment, the TMOF receptor is encoded by a polynucleotidesequence comprising the coding sequence (nucleotides I - 186) shown inSEQ ID NO. 3 or other polynucleotide sequence with codons encoding theamino acid sequence of SEQ ID NO. 4.

Isolated TMOF receptors can be used to produce antibodies according toknown techniques. These antibodies may be monoclonal or polyclonal, andcan be used to screen an expression library to identify other clonesexpressing polypeptides having TMOF receptor activity. Alternatively,these antibodies may be used to identify TMOF receptors from theirnatural material, such as mosquito or insect gut material.

A specific TMOF receptor sequence is exemplified herein. This sequenceis merely exemplary of TMOF receptors. Variant or equivalent receptors(and nucleotide sequences coding for equivalent receptors) having thesame or similar TMOF receptor activity can also be utilized. Equivalentreceptors will typically have amino acid homology with the exemplifiedreceptor. This amino acid identity will typically be greater than 60%,preferably be greater than 75%, more preferably greater than 80%, morepreferably greater than 90%, and can be greater than 95%. Theseidentities are determined using standard alignment techniques. The aminoacid homology will be highest in critical regions of the receptor whichaccount for biological activity or are involved in the determination ofthree- dimensional configuration which ultimately is responsible for thebiological activity. In this regard, certain amino acid substitutionsare acceptable and can be expected if these substitutions are in regionswhich are not critical to activity or are conservative amino acidsubstitutions which do not affect the three-dimensional configuration ofthe molecule. For example, amino acids may be placed in the followingclasses: non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby an amino acid of one class is replaced withanother amino acid of the same type fall within the scope of the subjectinvention so long as the substitution does not completely eliminate thebiological activity of the compound; however, preferred substitutionsare those which result in the retention of most or all of the biologicalactivity of the compound. Table 1 provides a listing of examples ofamino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not completelyeliminate the biological activity of the receptor; however, preferredsubstitutions are those which result in the retention of most or all ofthe biological activity of the compound.

The use of polynucleotide probes is well known to those skilled in theart. In one specific example, a cDNA library for mosquito or insect gutcells can be created by routine means, and DNA of interest can beisolated from the cDNA library. Polynucleotides of the subject inventioncan be used to hybridize with DNA fragments of the constructedcDNA-library, allowing identification of and selection (or “probingout”) of the genes of interest, i.e., those nucleotide sequences whichhybridize with the probes of the subject invention and encodepolypeptides having TMOF receptor activity. The isolation of these genescan be performed by a person skilled in the art having the benefit ofthe instant disclosure, using techniques which are well-known in themolecular biology art.

Thus, it is possible, without the aid of biological analysis, toidentify polynucleotide sequences encoding TMOF receptors. Such a probeanalysis provides a rapid method for identifying genes encoding TMOFreceptors from a wide variety of hosts. The isolated genes can beinserted into appropriate vehicles which can then be used to transform asuitable host.

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementarity that is requiredfor duplex formation. Severity of conditions can be controlled bytemperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under moderate tohigh stringency conditions by techniques well known in the art, asdescribed, for example, in Keller, G. H., M. M. Manak [1987] DNA Probes,Stockton Press, New York, N.Y., pp. 169-170.

Examples of various stringency conditions are provided herein.Hybridization of immobilized DNA on Southern blots with ³²P-labeledgene-specific probes can be performed by standard methods (Maniatis etal. [ 1982] Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York). In general, hybridization and subsequent washescan be carried out under moderate to high stringency conditions thatallow for detection of target sequences with homology to the exemplifiedpolynucleotide sequence. For double-stranded DNA gene probes,hybridization can be carried out overnight at 20-25±C below the meltingtemperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution,0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is describedby the following formula:

Tm=81.5° C.+16.6 Log[Na+]+0.41(%G+C)−0.61 (%formamide)−600/length ofduplex in base pairs

(Beltz et al. [1983] Methods of Enzymology, R. Wu, L. Grossman and K.Moldave [eds.] Academic Press, New York 100:266-285).

Washes are typically carried out as follows:

(1) twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash);

(2) once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20° C. below the melting temperature (Tm) of the hybrid in6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes can be determined by the following formula:

Tm (° C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs,S.V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B.Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D.Brown [ed.], Academic Press, New York, 23:683-693).

Washes can be carried out as follows:

(1) twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (lowstringency wash);

(2) once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment>70 or so bases in length, the followingconditions can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or 2×SSPE, 42° C.

Moderate: 0.2× or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

Identification of Pesticidal Polypeptides and other PesticidalCompounds. The TMOF receptors can advantageously be used to identifypesticidal polypeptides and other pesticidal compounds which activatethe TMOF receptor. As noted above, activation of the TMOF receptorinhibits biosynthesis of TTLE, thereby inhibiting digestion of proteinsand peptides and decreasing the availability of essential amino acids. Aperson skilled in the art, having the benefit of the instant disclosure,can utilize the TMOF receptors described herein to identify novelpesticidal polypeptides and other non-peptide pesticidal compounds. Inone embodiment, the TMOF receptor can be purified from its naturalsources using, for example, antibodies to the TMOF receptor to obtainthe purified protein. This purified protein can then be used to identifycompounds which bind to the receptor. Compounds thus identified can thenbe further evaluated using, for example, appropriate bioassays toconfirm and/or characterize the pest control activity of the compound.

As an alternative to purifying TMOF receptors from their naturalmaterial, recombinant TMOF receptor protein can be expressed in anappropriate recombinant host which has been transformed with apolynucleotide sequence encoding the TMOF receptor. The polynucleotidesequence used to transform the appropriate host may comprise, forexample, the polynucleotide coding sequence disclosed in SEQ ID NO. 3.The host may be transformed so as to express the TMOF receptor at thecell surface or, alternatively, the TMOF receptor may be retainedintracellularly or secreted into the surrounding media. In any case, theexpressed TMOF receptor may be isolated from the recombinant host usingtechniques known to those skilled in the art. The recombinant purifiedprotein can then be used as described above to identify compounds whichbind to the receptor. As an alternative embodiment, the receptorexpressed at the surface of the recombinant cell can be used inconjunction with the whole cell to identify compounds which bind to thereceptor.

In another embodiment, TMOF receptors of the subject invention can beapplied to a chip or other suitable substrate to facilitate highthroughput screening of potential pesticidal polypeptides and othernon-peptide pesticidal compounds.

Once compounds are identified which bind to the TMOF receptor, theirpesticidal activity can be confirmed and/or characterized usingbioassays known to those skilled in the art. The pesticidal polypeptidesand other pesticidal compounds of the subject invention can haveactivity against a variety of pests, for example, agricultural pestswhich attack plants as well as pests of animals which attack humans,agricultural animals, and/or domestic animals.

Production of recombinant hosts. The various methods employed in thepreparation of the plasmids and transformation of host organisms arewell known in the art; some of these methods are described in U.S. Pat.Nos. 5,011,909 and 5,130,253. These patents are incorporated herein byreference. Plasmid preparation procedures are also described in Maniatiset al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York. From these references and many others it can beseen that it is within the skill of those in the genetic engineering artto extract DNA from microbial cells, perform restrictions enzymedigestions, electrophorese DNA fragments, tail and anneal plasmid andinsert DNA, ligate DNA, transform cells, e.g., E. coli or plant cells,prepare plasmid DNA, electrophorese proteins, and sequence DNA.

Various markers may be employed for the selection of transformants,including biocide resistance, particularly to antibiotics such asampicillin, tetracycline, trimethoprim, chloramphenicol and penicillin;toxins, such as colicin; and heavy metals, such as mercuric salts.Alternatively, complementation (providing an essential nutrient to anauxotrophic host) may be employed.

The polynucleotide sequences of the subject invention can be introduceddirectly into the genome of the transformable host cell or can first beincorporated into a vector which is then introduced into the host.Exemplary methods of incorporation include transduction by recombinantphage or cosmids, transfection where specially treated host bacterialcells can be caused to take up naked phage chromosomes, andtransformation by calcium precipitation. These methods are well known inthe art. Exemplary vectors include plasmids, cosmids and phages.

It is well known in the art that when synthesizing a gene for improvedexpression in a host cell it is desirable to design the gene such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell. For purposes of the subject invention,“frequency of preferred codon usage” refers to the preference exhibitedby a specific host cell in usage of nucleotide codons to specify a givenamino acid. To determine the frequency of usage of a particular codon ina gene, the number of occurrences of that codon in the gene is dividedby the total number of occurrences of all codons specifying the sameamino acid in the gene. Similarly, the frequency of preferred codonusage exhibited by a host cell can be calculated by averaging frequencyof preferred codon usage in a large number of genes expressed by thehost cell. It is preferable to limit this analysis to genes that arehighly expressed by the host cell.

Thus, in one embodiment of the subject invention, cells can begenetically engineered, e.g., transformed with polynucleotides encodingthe subject peptides to attain desired expression levels of the subjectpeptides. To provide genes having enhanced expression, the DNA sequenceof the gene can be modified to comprise codons preferred by highlyexpressed genes to attain an A+T content in nucleotide base compositionwhich is substantially that found in the transformed host cell. It isalso preferable to form an initiation sequence optimal for the host celland to eliminate sequences that cause destabilization, inappropriatepolyadenylation, degradation and termination of RNA, and to avoidsequences that constitute secondary structure hairpins and RNA splicesites. For example, in synthetic genes, the codons used to specify agiven amino acid can be selected with regard to the distributionfrequency of codon usage employed in highly expressed genes in the hostcell to specify that amino acid. As is appreciated by those skilled inthe art, the distribution frequency of codon usage utilized in thesynthetic gene is a determinant of the level of expression.

Assembly of the polynucleotide sequences of this invention can beperformed using standard technology known in the art. For example, astructural gene designed for enhanced expression in a host cell can beassembled within a DNA vector from chemically synthesizedoligonucleotide duplex segments. Preferably, the DNA vector or constructhas an operable promoter and suitable termination signals. Thepolynucleotide sequence can then be introduced into a host cell andexpressed by means known in the art. Preferably, the pesticidalpolypeptide or receptor produced upon expression of the nucleotidesequence is functionally equivalent to the purified peptide. Accordingto the subject invention, “functionally equivalent” refers to retentionof function such as, for example, pest control activity.

Furthermore, chimeric polypeptides may be made and used according to thesubject invention. These chimeric polypeptides may comprise two or morepesticidal polypeptides of the present invention linked by peptidebonds, disulfide bonds or other chemical bonds known in the art forjoining amino acids, and may optionally include one or more heterologouspolypeptides joined in like manner to one or more pesticidalpolypeptides of the present invention. The portions that are combinedneed not be pesticidal, so long as the combination of portions creates achimeric protein which is pesticidal. The chimeric polypeptides mayinclude portions from toxins which do not necessarily act upon the TMOFreceptor. For example, toxins from Bacillus thuringiensis (B.t.)andtheir various toxin domains are well known to those skilled in the art;e.g., B.t. israeliensis, B.t. tenebrionis, B.t. san diego, B.t. aizawai,B.t. subtoxicus, B.t. alesti, B.t. gallaeriae, B.t. sotto, B.t.kurstaki, B.t. berliner, B.t. tolworthi, B.t. dendrolimus and B.t.thuringiensis, as well as B.t. toxins described in U.S. Pat. No.5,686,069, including various delta-endotoxins.

With the teachings provided herein, one skilled in the art can readilymake and use the various polypeptides and polynucleotide sequencesdescribed herein.

The polynucleotide sequences and pesticidal polypeptides usefulaccording to the subject invention include not only the exemplifiedsequences but also fragments of these sequences, variants, mutants, andfusion proteins which retain the characteristic pesticidal activity ofthe peptides specifically exemplified herein. As used herein, the terms“variants” or “variations” of genes refer to nucleotides havingdifferent nucleotide sequences encoding the same peptides or encodingequivalent peptides having pesticidal activity. As used herein, the term“equivalent peptides” refers to analogues, derivatives, fragments andother variants having the same or similar biological activity as theexemplified peptides which activity may be increased or reduced butwhich is not entirely eliminated.

Variations of genes may be readily constructed using standard techniquesfor making point mutations. Also, fragments of these genes can be madeusing commercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as BAL31 or site-directedmutagenesis can be used to systematically excise nucleotides from theends of these gene fragments. Also, genes encoding active fragments maybe obtained using a variety of restriction enzymes. Proteases may beused to directly obtain active fragments of these peptides.

Polynucleotide sequences encoding pesticidal polypeptides of the subjectinvention can be introduced into a wide variety of microbial or planthosts, such that expression of the gene results, directly or indirectly,in the production and maintenance of the pesticide. With suitablemicrobial hosts, e.g. yeast or Chlorella, the microbes can be applied tothe situs of the pest where they will proliferate and be ingested,resulting in control of the pest. Alternatively, the microbe hosting thegene can be killed and treated under conditions that retain and/orprolong the activity of the pesticidal polypeptide and stabilize thecell. The treated cell, which retains the toxic activity, can then beapplied to the environment of the target pest. In one embodiment, thehost is transformed such that the gene encoding the pesticidalpolypeptide is only expressed or maintained for a relatively shortperiod of time, such as days or weeks, so that the material does notpersist in the environment.

A wide variety of means are available for introducing a polynucleotidesequence encoding a pesticidal polypeptide into a microorganism hostunder conditions which allow for stable maintenance and expression ofthe gene. These methods are well known to those skilled in the art andare described, for example, in U.S. Pat. No. 5,135,867, which isincorporated herein by reference.

Synthetic genes encoding peptides that are functionally equivalent tothe pesticidal polypeptides of the subject invention can also be used totransform hosts. Methods for the production of synthetic genes can befound for example, in U.S. Pat. No. 5,380,831.

Recombinant cells expressing a pesticidal polypeptide can be treated toprolong the pesticidal activity of the polypeptide and optionally tostabilize the cell. Pesticide microcapsules can be formed which comprisethe pesticidal polypeptide within a stabilized cellular structure andprotect the pesticidal polypeptide when the microcapsule is applied tothe environment of the target pest. Suitable host cells includeprokaryotes and eukaryotes. Preferred hosts include prokaryotes andlower eukaryotes, such as algae and fungi. The cell is preferably intactand substantially in the proliferative form when treated, rather than ina spore form.

Treatment of the microbial cell, e.g., a microbe comprising thepolynucleotide sequence encoding the pesticidalpolypeptide, can be bychemical or physical means, or by a combination of chemical and/orphysical means, so long as the technique does not completely eliminatethe pesticidal properties of the pesticidal polypeptides and does notdiminish the cellular capability of protecting the pesticidalpolypeptide. Methods for treatment of microbial cells are disclosed inU.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein byreference.

Formulations and Administration. As would be appreciated by a personskilled in the art, the pesticidal concentration will vary widelydepending upon the nature of the particular formulation, particularlywhether it is a concentrate or to be used directly. The pesticide willbe present in at least about 0.0001% by weight and may be 100% byweight. The dry formulations will have from about 0.0001-95% by weightof the pesticide while the liquid formulations will generally be fromabout 0.0001-60% by weight of the solids in the liquid phase. Theformulations that contain cells will generally have from about 1 toabout 10¹⁰ cells/mg. These formulations will be administered at about 50mg (liquid or dry) to 1 kg or more per hectare.

In applications to the environment of the target pest, the transformantstrain can be applied to the natural habitat of the pest in a living ornon-living state. When applied in a living state, the transformantstrain may grow in the pest upon ingestion while producing thepesticidal polypeptide, which will have a deleterious effect on thepest. The organism may be applied by pouring, spraying, soaking,injection into the soil, seed coating, seedling coating or spraying, orthe like.

In aquatic environments, pest control may be attained at or below thesurface by adjusting the specific gravity of the microbe. Where thepesticidal polypeptide is applied as a component of a transformantmicrobe, depth control may be accomplished by varying the lipid contentof the transformant microorganism strain. It is known that indigenousaquatic algae float due to their lipid content. A variation in lipidcontent will allow the transformant strain to be distributed at desireddepths below the water surface.

Pesticidal polypeptides may also be formulated as tablets, pellets,briquettes, bricks or the like, which maintain the pesticidalpolypeptide at a specific depth in an aqueous environment. In oneembodiment, the compositions of the present invention are formulated tofloat on the surface of an aqueous medium; in another embodiment, theyare formulated to maintain a depth of 0 to 2 feet in an aqueous medium;in yet another embodiment, the compositions are formulated to sink in anaqueous medium.

For commercial formulations, the organisms may be maintained in anutrient medium (e.g., yeast extract or L-broth) which maintainsselectivity and results in a low rate of proliferation. To prepare theorganism for application, the non-proliferating concentrate may beintroduced into an appropriate selective nutrient medium, grown to highconcentration, generally from about 1 to 10⁹ cells/ml and may then beemployed for introduction into the environment of the pest.

All of the U.S. patents and other references cited herein are herebyincorporated by reference, as are U.S. patent application Ser. No.09/295,849, (UF-216) “Neuropeptides and their use for Pest Control”;U.S. patent application Ser. No. 09/296,113, (UF-224) “Materials andMethods Useful for the Control of Insect Larvae”; U.S. patentapplication Ser. No. 09/295,996, (UF-230) “Novel Peptides and the UseThereof to Control Pests”; and U.S. patent application Ser. No.09/295,924, (IPTL Docket No. 4137-120) “Compositions and Methods forControlling Pests”.

Materials and Methods

Chlorella Strain. Chlorella isolated from an irrigation canal near Ft.Pierce, Florida was provided by Dr. Charles Powell of the University ofFlorida-IFAS, Indian River Research Education and Agricultural Station,Ft. Pierce, FL. For other tranformations, Chlorella dessiccata was used.

Plasmid Strain. In some procedures Chlorella was transformed withpKylx71, from Dr. Arthur Hunt of the University of Kentucky, Departmentof Agronomy, Lexington, Kentucky. In other procedures, transformationwas achieved in the salt water Chlorella desiccata using the followingplasmids that carry TMOF: pYES/TMOF, pYDB3 and pYDB4 (FIGS. 8-10).

Green Fluorescent Protein TMOF Gene. Single strand oligonucleotidesprimers containing the GFP and TMOF gene sequences were designed asfollows:

Forward Primer:

5′ AAGGTACCATGGCTAGCAAAGGAGAAGAA 3′ (DB 207) (SEQ ID NO. 5)

and Backward Primer:

5′ TTTCTAGATCAAGGAGGAGGAGGAGGAGGTGCTGGATCATATCTACCTTCGATTTTGTAGAGCTCATCCAT 3′ (DB 209) (SEQ ID NO. 6)

The forward primer carried a Kpn I restriction site, and the backwardprimer carried a Xba I restriction site. To bridge between the Xho Irestriction site on the plasmid and the Kpn I site on the forward primera third oligonucleotide (5′ TCGAGGGTAC 3′) (DB 208) (SEQ ID NO. 7) wassynthesized. The GFP-TMOF gene was designed in such a way that itcarried an ATG start signal and a TGA stop signal and a trypsin cleavagesite sequence (IEGR). The forward and backward primers were used toamplify a GFP-TMOF dsDNA (800 bp) from 30B Cycle 3 GFP, 10 Kbp plasmid.The GFP-TMOF gene was cut with Kpn I and Xba I and directionally ligatedinto a pKylx71 that was cut with Xba I and Xho I, in the presence of anoligonucleotides bridge (DB 208).

Transformation of Chlorella. Chlorella cells were grown overnight,concentrated by centrifugation and incubated with 1% cellulase and 0.1%pectinase for 1 hour to partially digest the cell wall. TreatedChlorella cells were incubated with pKylx 71 GFP-TMOF plasmid in thepresence of polyethylene glycol and the cells were grown on MBBM agarplates in the presence of Kanamycin (20 μg/ml) for several days at roomtemperature under light. Several colonies were picked with a sterileloop from each plate and transferred to tissue culture bottlescontaining 10 ml of MBBM media and Kanamycin and grown for several daysto cell densities of 10⁸ cell/ml. Aliquots from each tissue culture wereassayed for TMOF biosynthesis by ELISA (3±0.1 μg TMOF was synthesizedper ml) and for larvicidal activity by adding 30 μl aliquots (90±3 ng ofrecombinant GFP-TMOF; 3×10⁴ cells) into 100 μl of distilled water inmicrotiter plates. Each well contained a single 2nd instar larvae ofAedes aegypti. Control wells contained yeast cells or non-transformedChlorella cells. Larval growth and mortality was checked every 24 hoursunder a dissecting microscope. Within 72 hours, 95% of the larvae thatwere fed on GFP-TMOF Chlorella died, whereas 96% and 90% of the larvaethat were fed normal Chlorella and yeast cells, respectively, survived.The control larvae eventually pupated and normal adults emerged. Theseresults demonstrate that Chlorella GFP-TMOF can be used as an effectivemosquito larvicide. Because the GFP-TMOF gene did not incorporate intothe Chlorella genome, the synthesis of GFP-TMOF is transient and thegene would not stay in the water long enough to induce resistance in thelarvae.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Transformation of Yeast with pYDB2

A plasmid for the transformation of yeast cells was designed and namedpYDB2 (FIG. 1). The jellyfish Green Fluorescent Protein (GFP) and TMOFwere cloned in tandem as a fusion protein with a trypsin cleavage sitein the multiple cloning site of this plasmid (FIG. 1). The plasmid wasintegrated into the genome of the brewer's yeast Saccharomycescerevisiae by homologous recombination and the GFP-TMOF fusion proteinwas expressed by the yeast.

To achieve homologous recombination of the fusion protein GFP-TMOF intothe yeast genome, plasmid pYES2 (Invitrogen, CA) was modified. TheInvitrogen plasmid was not designed to integrate into the yeast's genomebut to express proteins as a high copy plasmid. To convert the plasmidfrom an autonomous replicating entity to a plasmid that can be used forhomologous recombination the following changes and modifications weredone:

a. The plasmid DNA was cut with two restriction enzymes Nael at position1007 and ClaI at position 2873 and the plasmid was recircularized andnamed pYDB2 (FIG. 1).

b. The multiple cloning site was then cut with two restriction enzymesXbaI and KpnI and the opened plasmid was ligated at the XbaI and KpnIrestriction sites with a TMOF-GFP DNA containing a trypsin cleavage sitesequence (IEGR, see FIG. 1). The new plasmid (4.9 Kb) was named pYDB2GFP-TMOF (FIG. 1).

c. The plasmid was opened with ApaI and introduced into yeast cells withURA3 mutation using Lithium Acetate. The transformed yeast cells weregrown on synthetic drop out medium lacking Uracil (SD-URA) to select forcolonies that carry the URA3 gene and GFP-TMOF (FIGS. 2A and 2B).

Colonies were grown on SD-URA medium for 48 hours in the presence ofraffinose and induced with galactose for 1 to 8 hours. TMOF biosynthesiswas analyzed by ELISA (FIG. 3) and GFP by fluorescence microscopy.

EXAMPLE 2 Control of Mosquito Larvae with Recombinant Yeast GFP-TMOF

Aedes aegypti larvae were fed individually in microtiter platescontaining 1.0 ml of water and 1.5×10⁶ cells. At 24 hour intervals theplates were examined under a microscope and dead larvae were counted.The experiment was repeated 6 times with groups of 24 larvae using liveyeast cells, or heat inactivated cells. In both cases 95% of themosquito larvae died within 144 hours (FIGS. 4 and 5). Those larvae thatdid not die, did not grow. On the other hand, 100% of the controls thatwere fed on live yeast, or heat inactivated cells were alive and thelarvae grew to the pupal stage and the newly emerged adults were normal,i.e., they took a blood meal and produced a clutch of eggs. Similarresults were obtained when 20 larvae were added to containers with 100ml of water and 6×10⁸ cells. All the larvae that fed on recombinantyeast died within 6 to 7 days, whereas controls grew to the pupal stageand the adults that emerged from the pupae were normal.

EXAMPLE 3 Transformation of Chlorella with pKvlx71

The GFP-TMOF gene from Example 1 was cloned into pKylx7l at the multiplecloning site (FIG. 6) and pKylx71 carrying GFP-TMOF was inserted intoChlorella in the presence of polyethylene glycol. Non-transformedChlorella are not Kanamycin resistant. Transformed algae were grown onagar plates in the presence of Kanamycin to select for resistance.Single colonies were removed from the agar plate and grown in liquidmedia in the presence of Kanamycin and under constant light. The newlygrown Chlorella cells were analyzed for GFP-TMOF synthesis by ELISA.Chlorella cells that produced TMOF were then fed to mosquito larvae.

Larval survival and development were compared with controls that werefed on Chlorella cells that did not produce GFP-TMOF or on normal yeastcells (FIG. 7). Ninety-five percent of the larvae that were fed onGFP-TMOF died within 3 days. Larvae that did not die did not grow anddid not reach the adult stage. Larvae that were fed normal Chlorella(96%) or yeast cells (90%) developed into normal adults (FIG. 7). Theuse of Chlorella as the host organism for delivering the pesticidalpolypeptides to mosquito larvae is advantageous since Chlorellanaturally inhabit the niche occupied by mosquito larvae and are anatural food of mosquito larvae.

Chlorella is a particularly attractive vehicle for expressing TMOF formosquito control. Chlorella is ubiquitously present worldwide andreadily grows under conditions where mosquito larvae also grow. In fact,Chlorella are a natural food source for mosquito larvae. Chlorella arereadily susceptible to genetic manipulation using well establishedcloning vectors and techniques. Furthermore, high levels of expressionof cloned proteins and peptides can be obtained. Releasing the algaeinto the water does not upset the ecological balance because algaegrowth is naturally controlled by several viruses that prevent rapidgrowth (blooming).

EXAMPLE 4 Transformation of Chlorella desiccata

Although pYES/TMOF does not carry a homologous algal gene for homologousrecombination, several reports in the literature indicate that the yeast2 μ origin of replication either integrates into the genome ofChlamydomonas reinhardtii or causes autonomous replication in the algae(Rochaix et al., 1988). Plasmids pYDB3 and pYDB4 carry 2.3 and 8.0 Kbfragments of the nitrate reductase gene from Chlorella vulgaris (Dawsonet al., 1996), respectively, and were used for homologous recombinationin Chlorella desiccata. All the plasmids express TMOF behind a strongpromoter of the cauliflower Tobacco Mosaic (CaMV) 35S². The TMOFsynthetic gene was designed so it could be directionally cloned intopKylx 71 at the Xho I and Aba I sites. The TMOF synthetic gene carriesan ATG start signal and a TGA stop signal as follows (SEQ ID NOs. 65 and66):

   Xho I  Start                          Stop  XbaI5′ TCGAGATGTATGATCCAGCACCTCCTCCTCCTCCTCCTTGAT       CTACATACTAGGTCGTGGAGGAGGAGGAGGAGGAACTAGATC

The outer wall of Chlorella desiccata was removed with cellulase andpectinase and Chlorella desiccata was transformed with pYES/TMOF, pYDB3and pYDB4 in the presence of polyethanollglycol. After transformation,algal cells were grown on artificial salt-water agar in the presence ofGeneticin (600 μg/ml) to select for resistance. Non transformedChlorella desiccata does not grow and dies in the presence of Geneticin(400-600 μg/ml). Single colonies were removed from the agar plate andgrown in salt-water liquid media in the presence of Geneticin (400μg/ml) and under constant light. Chlorella cells were analyzed forGFP_TMOF synthesis by ELISA. The recombinant cells synthesized about 2to 3.7 μg of TMOF per 107 cells. Chlorella cells that produced TMOF werefed to mosquito larvae and larval survival and development was comparedwith controls that were fed on Chlorella cells that: a) carried theplasmids without the TMOF gene, b) cells that did not carry any plasmid,and c) normal yeast cells (Table 2).

TABLE 2 Feeding of Aedes aegypti larvae with recombinant Chlorelladesiccata Cell type Groups Mortality (%) ± S.E.M. a. pYES2/TMOF+ 3 100 ±0 b. Yeast 3  0 c. pYES2/TMOF− 3  0 d. pKylx/TMOF+ 3 100 ± 0 e.pKylx/TMOF− 3  0 f. pYDB3 3 100 ± 0 g. pYDB4 3 100 ± 0 h. Chlorelladesiccata 3  7 ± 0.8

Groups ofAedes aegypti larvae (10 per group) were fed for 3-9 days withtransformed and nontransformed Chlorella desiccata (10⁶ cells). Controlswere nontransformed Chlorella or yeast cells (10⁶ cells). Results are anaverage of3 determinations±S.E.M. In a, d, f, and g 90% mortality wasobserved in 3 days.

The advantage of using this system is that mosquito larvae eat Chlorellain the wild and being an aquatic organism the Chlorella persists longerin the water and provides an economical means to control mosquitopopulations in the marsh.

EXAMPLE 5 Effect of TMOF Analogues on Mosquito Larvae

TMOF can traverse the gut epithelium, enter the hemolymph and bind a gutreceptor (Borovsky, D. and F. Mahmood (1995) “Feeding the mosquito Aedesaegypti with TMOF and its analogs; effect on trypsin biosynthesis andegg development,” Regulatory Peptides57:273-281; Borovsky, et al. (1994)“Characterization and localization of mosquito-gut receptors for trypsinmodulating oostatic factor using complementary peptide immunochemistry”FASEB J. 8:350-355.). These results allowed the development of atechnique by which TMOF and its analogues can directly be tested byfeeding them to mosquito and other pest larvae. To find out if truncatedTMOF peptides have an effect on larval growth and development, a seriesof peptides were synthesized and tested by feeding them to mosquitolarvae at concentrations of 0 to 5.0 mg/ml (Table 3). Individual, newlyhatched Aedes aegypti larvae were maintained in separate microtiterwells on a diet of autoclaved yeast (1 mg/ml). The diet was supplementedwith TMOF peptides (Table 3). An identical number of larvae maintainedon yeast served as a control. Larvae that were fed on differentconcentrations of TMOF peptides (0 mg/ml to 5.0 mg/ml) were monitoredfor eight (8) days for survival and larval growth and development. Allcontrol groups survived and larval growth and development was normal.Since larvae swallow only a small portion of the yeast particles thatadsorbed the peptides, it is assumed that approximately 1 to 20 ng aretaken orally at the high concentrations. These results allowed thecalculation of the Lethal Dose at 50% mortality (LD₅₀; Table 3) of theTMOF peptides.

TABLE 3 The Effect of TMOF and its analogue peptides on mosquito larvaeSEQ ID LD₅₀ Compound NO: N mM ± S.E.M.  1. YDPAP₆ 11 3  0.2 ± 0.02  2.MPDYP₅ 32 3 >3.0  3. YDPAF 47 3 0.33 ± 0.2  4. YEPAP 53 3 0.35 ± 0.02 5. FDPAP 30 3 0.37 ± 0.15  6. YDPLP 52 3  1.5 ± 0.04  7. YDPAL 48 30.52 ± 0.03  8. YAPAP 57 3 0.54 ± 0.13  9. YNPAP 55 3 0.55 ± 0.03 10.(D)YDPAP 49 3 0.56 ± 0.03 11. YFPAP 54 3 0.64 ± 0.03 12. YDPAP 14 3 1.64± 0.03 13. YDLAP 44 3  0.6 ± 0.05 14. YDFAP 42 3 0.74 ± 0.13 15. YDAAP40 3  1.0 ± 0.18 16. YDPGP 51 5  1.1 ± 0.18 17. Y(D)DPAP 36 3  1.2 ± 0.318. YSPAP 58 3  1.4 ± 0.03 19. YDPAA 59 3  1.6 ± 0.13 20. YDPFP 50 4 1.7 ± 0.4 21. ADPAP 21 4  2.0 ± 0.36 22. Y(D)DP 35 3 0.28 ± 0.01 23.DPA 25 3  0.4 ± 0.03 24. (D)YDP 46 3 0.51 ± 0.05 25. DAA 23 3 0.91 ±0.06 26. YDG 43 3 0.95 ± 0.11 27. YDF 41 3 0.97 ± 0.11 28. APA 22 3  1.0± 0.07 29. AAP 19 3 1.08 ± 0.07 30. YSF 56 3 1.08 ± 0.12 31. DYP 27 41.27 ± 0.17 32. YDA 39 3  1.6 ± 0.13 33. FDP 29 3 1.98 ± 0.6 34. YDP 455  2.3 ± 0.4 35. FSP 31 3  2.3 ± 0.13 36. YAP 37 3  2.3 ± 0.5 37. PAA 333  2.4 ± 0.34 38. PAP 34 3 3.17 ± 0.14 39. FAP 28 3  3.8 ± 0.23 40. ADP20 3 >6.6 41. YD 38 3 1.24 ± 0.06 42. DY 26 3  3.0 ± 0.8 Groups of 12 to24 mosquito larvae were incubated with different concentrations of TMOFand its analogue peptides in 100 μl microtiter plates for 7 days.Results are expressed as LD₅₀ ± S.E.M.

EXAMPLE 6 Control of Mosquito Larvae with Polypeptides ComprisingArginine

Individual, newly hatched Aedes aegypti larvae were maintained inseparate microtiter wells on a diet of autoclaved yeast (1 mg/ml). Thediet was supplemented with TMOF analogues (Table 4). An identical numberof larvae were maintained on yeast served as a control. Larvae that werefed on different concentrations of TMOF analogues (0.5 mg/ml to 5.0mg/ml) were monitored for 8 days for survival and larval growth anddevelopment. All control groups survived and larval growth anddevelopment were normal. Larvae that were fed TMOF analogues hadincreased mortality, particularly when the larvae where fed theconcentrations of the analogues higher than 2 mg/ml (Table 4).

TABLE 4 Effect of Feeding TMOF-R Analogues on Aedes aegypti LarvaeCompound SEQ ID NO: N LC₅₀ (mM ± S.E.M.) 1. YDPAP₆ 11 3  0.2 ± 0.02 2.YDPAP₆R 67 3  71.6>>>>> 3. YDPAPR 68 3  0.24 ± 0.02 4. YDPAFR 69 3  0.15± 0.023 5. YDPAR 70 3  0.12 ± 0.07 6. YDPR 71 3  0.24 ± 0.026 7. DPAR 603  0.46 ± 0.014 8. (DPAR)₄ 61 3 0.048 ± 0.002 Groups of Aedes aegypti(12 per group) were fed TMOF and TMOF-R analogues in microtiter plateswith yeast particles for 5 to 6 days. Larval mortality was checkeddaily. The results are the average of 3 determinations ± S.E.M.

Since larvae swallow only a small portion of the yeast particles thatadsorbed the analogues it is assumed that approximately I to 20 ng aretaken orally at the high concentrations. Using the variousconcentrations a lethal concentration that caused 50% mortality (LC₅₀)for the mosquito larvae was calculated (Table 4). The results presentedin Table 4 clearly indicate that the short TMOF-R analogues are veryeffective, especially the (DPAR)₄ (SEQ ID NO. 61) polymer that is brokeninto 4 DPARs (SEQ ID NO. 60) in the gut.

EXAMPLE 7 Injecting TMOF-r Analogues Into Heliothis virescens

Individual second instar larvae of H. virescens were injected withTMOF-R analogues (10 to 0.25 μg per larva) and maintained in separateplastic cups on artificial diet. Twenty-four hours after the injections3 groups of larvae (3 per group) were assayed for trypsin biosynthesisusing BApNA (trypsin specific substrate; Table 5).

TABLE 5 Effect of TMOF-R analogues on trypsin biosynthesis in Heliothisvirescens Compound SEQ ID NO: N Amount (μg/injections) Inhibition (% ±S.E.M.)  1. YDPAP₆ 11 3 10 56 ± 26  2. YDPAP₆ 11 3 1 25 ± 2   3. YDPAP₆11 3 0.5 11 ± 1   4. YDPAP₆ 11 3 0.25 19.5 ± 0.7   5. YDPAPR 68 3 10 53± 25  6. YDPAPR 68 3 1 31.5 ± 2    7. YDPAPR 68 3 0.5 14 ± 1   8. YDPAPR68 3 0.25 11 ± 1   9. YDPAFR 69 3 10 0 ± 0 10. YDPAR 70 3 10 33.5 ± 9  11. YDPAR 70 3 1 39 ± 2  12. YDPAR 70 3 0.5   1 ± 0.07 13. DPAR 60 3 10100 14. DPAR 60 3 1 17 ± 2  15. (DPAR)₄ 61 3 10 58 ± 36 16. (DPAR)₄ 61 31 35 ± 7  17. (DPAR)₄ 61 3 0.5   3 ± 0.3 Groups of H. virescens wereinjected with TMOF-R analogues in 0.5 μl of Sterile water and 24 hourslater trypsin biosynthesis was followed using BApNA. Results werecompared to controls that were injected with sterile distilled water andare expressed as an average of 3 determination ± S.E.M>

Trypsin biosynthesis was clearly inhibited 24 hours after injecting theTMOF-R analogues (Table 5). DPAR (SEQ ID NO. 60) at 10 μg inhibited 75%of trypsin biosynthesis, whereas TMOF caused 56% inhibition. Theseresults indicate that TMOF-like compounds control trypsin biosynthesisin H. virescens as was shown in mosquito, and that these analogues canbe used to control these agricultural pests.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

71 1 10 PRT Leptinotarsa decemlineata MOD_RES (10)..(10) AMIDATION 1 AlaArg Gly Pro Gln Leu Arg Leu Arg Phe 1 5 10 2 8 PRT Leptinotarsadecemlineata MOD_RES (8)..(8) AMIDATION 2 Ala Pro Ser Leu Arg Leu ArgPhe 1 5 3 378 DNA Aedes aegypti CDS (1)..(189) 3 ata ctg ggg agg ggg gggggg gac att ggg tta ctc agt tca gac caa 48 Ile Leu Gly Arg Gly Gly GlyAsp Ile Gly Leu Leu Ser Ser Asp Gln 1 5 10 15 agg agt ttc agc act gaaact ctg ctt aaa gaa cta aaa aga gaa gcg 96 Arg Ser Phe Ser Thr Glu ThrLeu Leu Lys Glu Leu Lys Arg Glu Ala 20 25 30 gcg gct gag gag cgg agt gctgcc tcc aac tcg ggg tcg gtg gtt ccc 144 Ala Ala Glu Glu Arg Ser Ala AlaSer Asn Ser Gly Ser Val Val Pro 35 40 45 ctc tcg gag caa agg ctg atg ggacat ctg gcg gcc gcg ctg tga 189 Leu Ser Glu Gln Arg Leu Met Gly His LeuAla Ala Ala Leu 50 55 60 gccggctttc ctgctgccac tttgggcgcc ttggatggagatcccaattg cagtttgtat 249 tttatttttt tataagggac acgtggaaaa accaaaccaaaccaaacaaa gccaacaaac 309 cacgacggtc cttattttaa acctcagact ccataaagaaacctttctat ccaaaaaaaa 369 aaaaaaaaa 378 4 62 PRT Aedes aegypti 4 Ile LeuGly Arg Gly Gly Gly Asp Ile Gly Leu Leu Ser Ser Asp Gln 1 5 10 15 ArgSer Phe Ser Thr Glu Thr Leu Leu Lys Glu Leu Lys Arg Glu Ala 20 25 30 AlaAla Glu Glu Arg Ser Ala Ala Ser Asn Ser Gly Ser Val Val Pro 35 40 45 LeuSer Glu Gln Arg Leu Met Gly His Leu Ala Ala Ala Leu 50 55 60 5 29 DNAArtificial Sequence Forward Primer (DB207) 5 aaggtaccat ggctagcaaaggagaagaa 29 6 71 DNA Artificial Sequence Backward Primer (DB209) 6tttctagatc aaggaggagg aggaggaggt gctggatcat atctaccttc gattttgtag 60agctcatcca t 71 7 10 DNA Artificial Sequence Oligonucleotide (DB 208) 7tcgagggtac 10 8 10 PRT Artificial Sequence TMOF peptide 8 Xaa Xaa XaaXaa Xaa Pro Pro Pro Pro Pro 1 5 10 9 4 PRT Artificial Sequence Flankingregion 9 Pro Pro Pro Pro 1 10 5 PRT Artificial Sequence Flanking region10 Pro Pro Pro Pro Pro 1 5 11 10 PRT Aedes aegypti 11 Tyr Asp Pro AlaPro Pro Pro Pro Pro Pro 1 5 10 12 10 PRT Artificial Sequence TMOFpeptide 12 Asp Tyr Pro Ala Pro Pro Pro Pro Pro Pro 1 5 10 13 8 PRTArtificial Sequence TMOF peptide 13 Pro Ala Pro Pro Pro Pro Pro Pro 1 514 5 PRT Artificial Sequence TMOF peptide 14 Tyr Asp Pro Ala Pro 1 5 156 PRT Artificial Sequence TMOF peptide 15 Tyr Asp Pro Ala Pro Pro 1 5 167 PRT Artificial Sequence TMOF peptide 16 Tyr Asp Pro Ala Pro Pro Pro 15 17 8 PRT Artificial Sequence TMOF peptide 17 Tyr Asp Pro Ala Pro ProPro Pro 1 5 18 6 PRT Artificial Sequence TMOF peptide 18 Asn Pro Thr AsnLeu His 1 5 19 3 PRT Artificial Sequence TMOF peptide 19 Ala Ala Pro 120 3 PRT Artificial Sequence TMOF peptide 20 Ala Asp Pro 1 21 5 PRTArtificial Sequence TMOF peptide 21 Ala Asp Pro Ala Pro 1 5 22 3 PRTArtificial Sequence TMOF peptide 22 Ala Pro Ala 1 23 3 PRT ArtificialSequence TMOF peptide 23 Asp Ala Ala 1 24 2 PRT Artificial Sequence TMOFpeptide 24 Asp Phe 1 25 3 PRT Artificial Sequence TMOF peptide 25 AspPro Ala 1 26 2 PRT Artificial Sequence TMOF peptide 26 Asp Tyr 1 27 3PRT Artificial Sequence TMOF peptide 27 Asp Tyr Pro 1 28 3 PRTArtificial Sequence TMOF peptide 28 Phe Ala Pro 1 29 3 PRT ArtificialSequence TMOF peptide 29 Phe Asp Pro 1 30 5 PRT Artificial Sequence TMOFpeptide 30 Phe Asp Pro Ala Pro 1 5 31 3 PRT Artificial Sequence TMOFpeptide 31 Phe Ser Pro 1 32 9 PRT Artificial Sequence TMOF peptide 32Met Pro Asp Tyr Pro Pro Pro Pro Pro 1 5 33 3 PRT Artificial SequenceTMOF peptide 33 Pro Ala Ala 1 34 3 PRT Artificial Sequence TMOF peptide34 Pro Ala Pro 1 35 3 PRT Artificial Sequence TMOF peptide 35 Tyr XaaPro 1 36 5 PRT Artificial Sequence TMOF peptide 36 Tyr Xaa Pro Ala Pro 15 37 3 PRT Artificial Sequence TMOF peptide 37 Tyr Ala Pro 1 38 2 PRTArtificial Sequence TMOF peptide 38 Tyr Asp 1 39 3 PRT ArtificialSequence TMOF peptide 39 Tyr Asp Ala 1 40 5 PRT Artificial Sequence TMOFpeptide 40 Tyr Asp Ala Ala Pro 1 5 41 3 PRT Artificial Sequence TMOFpeptide 41 Tyr Asp Phe 1 42 5 PRT Artificial Sequence TMOF peptide 42Tyr Asp Phe Ala Pro 1 5 43 3 PRT Artificial Sequence TMOF peptide 43 TyrAsp Gly 1 44 5 PRT Artificial Sequence TMOF peptide 44 Tyr Asp Leu AlaPro 1 5 45 3 PRT Artificial Sequence TMOF peptide 45 Tyr Asp Pro 1 46 3PRT Artificial Sequence TMOF peptide 46 Xaa Asp Pro 1 47 5 PRTArtificial Sequence TMOF peptide 47 Tyr Asp Pro Ala Phe 1 5 48 5 PRTArtificial Sequence TMOF peptide 48 Tyr Asp Pro Ala Leu 1 5 49 5 PRTArtificial Sequence TMOF peptide 49 Xaa Asp Pro Ala Pro 1 5 50 5 PRTArtificial Sequence TMOF peptide 50 Tyr Asp Pro Phe Pro 1 5 51 5 PRTArtificial Sequence TMOF peptide 51 Tyr Asp Pro Gly Pro 1 5 52 5 PRTArtificial Sequence TMOF peptide 52 Tyr Asp Pro Leu Pro 1 5 53 5 PRTArtificial Sequence TMOF peptide 53 Tyr Glu Pro Ala Pro 1 5 54 5 PRTArtificial Sequence TMOF peptide 54 Tyr Phe Pro Ala Pro 1 5 55 5 PRTArtificial Sequence TMOF peptide 55 Tyr Asn Pro Ala Pro 1 5 56 3 PRTArtificial Sequence TMOF peptide 56 Tyr Ser Phe 1 57 5 PRT ArtificialSequence TMOF peptide 57 Tyr Ala Pro Ala Pro 1 5 58 5 PRT ArtificialSequence TMOF peptide 58 Tyr Ser Pro Ala Pro 1 5 59 5 PRT ArtificialSequence TMOF peptide 59 Tyr Asp Pro Ala Ala 1 5 60 4 PRT ArtificialSequence TMOF-R analogue peptide 60 Asp Pro Ala Arg 1 61 16 PRTArtificial Sequence TMOF-R analogue peptide 61 Asp Pro Ala Arg Asp ProAla Arg Asp Pro Ala Arg Asp Pro Ala Arg 1 5 10 15 62 2 PRT ArtificialSequence TMOF peptide 62 Xaa Xaa 1 63 10 PRT Artificial SequenceUnamidated version of NPF I 63 Ala Arg Gly Pro Gln Leu Arg Leu Arg Phe 15 10 64 8 PRT Artificial Sequence Unamidated version of NPF II 64 AlaPro Ser Leu Arg Leu Arg Phe 1 5 65 42 DNA Artificial Sequence TMOFsynthetic gene sense strand 65 tcgagatgta tgatccagca cctcctcctcctcctccttg at 42 66 42 DNA Artificial Sequence TMOF synthetic geneantisense strand 66 ctagatcaag gaggaggagg aggaggtgct ggatcataca tc 42 6711 PRT Artificial Sequence TMOF-R analogue peptide 67 Tyr Asp Pro AlaPro Pro Pro Pro Pro Pro Arg 1 5 10 68 6 PRT Artificial Sequence TMOF-Ranalogue peptide 68 Tyr Asp Pro Ala Pro Arg 1 5 69 6 PRT ArtificialSequence TMOF-R analogue peptide 69 Tyr Asp Pro Ala Phe Arg 1 5 70 5 PRTArtificial Sequence TMOF-R analogue peptide 70 Tyr Asp Pro Ala Arg 1 571 4 PRT Artificial Sequence TMOF-R analogue peptide 71 Tyr Asp Pro Arg1

What is claimed is:
 1. A recombinant host transformed with apolynucleotide encoding a pesticidal polypeptide, wherein saidpolypeptide comprises an amino acid sequence having the general formula:A¹A²A³A⁴A⁵Fl(Formula I) (SEQ ID NO. 8) wherein: A¹ is selected from thegroup consisting of A, D, F, G, M, P, S and Y; A² is selected from thegroup consisting of A, D, E, F, G, N, P, S and Y; A³ is optionallypresent and is selected from the group consisting of A, D, F, G, L, P,R, S and Y; A⁴ is optionally present when A³ is.present and is selectedfrom the group consisting of A, F, G, L, R; and Y; A⁵ is optionallypresent when A⁴ is present and is selected from the group consisting ofA, F, L, P and R; and Fl is a flanking region which is optionallypresent and is selected from the group consisting of: P, PP, PPP, PPPP(SEQ ID NO. 9), and PPPPP (SEQ ID NO. 10); with the proviso that thepolypeptide does not consist of YDPAP₆(SEQ ID NO. 11), DYPAP₆ (SEQ IDNO. 12), PAP₆ (SEQ ID NO. 13), YDPAP (SEQ ID NO. 14), YDPAP2 (SEQ ID NO.15), YDPAP₃ (SEQ ID NO. 16), YDPAP₄ (SEQ ID NO. 17), NPTNLH (SEQ ID NO.18) or DF-OMe.
 2. The transformed host, according to claim 1, whereinonly A¹, A², A³, A⁴ and Fl are present in the formula.
 3. Thetransformed host, according to claim 1, wherein only A¹, A², A³ and A⁴are present in the formula.
 4. The transformed host, according to claim1, wherein only A¹, A², A³ and Fl are present in the formula.
 5. Thetransformed host, according to claim, 1, wherein only A¹. A² and A³ arepresent in the formula.
 6. The transformed host, according to claim 1,wherein only A¹, A² and Fl are present in the formula.
 7. Thetransformed host, according to claim 1, wherein only A¹ and A² arepresent in the formula.
 8. The transformed host, according to claim 1,wherein A¹ is selected from the group consisting of A, D, F, M and Y,and A² is selected from the group consisting of A, D, E, P and Y.
 9. Thetransformed host, according to claim 1, wherein the polypeptide sequencecomprises A, D and Y.
 10. The transformed host, according to claim 1,wherein said polypeptide comprises A and D.
 11. The transformed host,according to claim 1, wherein the polypeptide has from 2 to 5 aminoacids.
 12. The transformed host, according to claim 1, wherein theN-terminus of the polypeptide is acetylated or the C-terminus of thepolypeptide is amidated, or both.
 13. The transformed host, according toclaim 1, wherein the C-terminus of said polypeptide is an arginine. 14.The transformed host, according to claim 1, wherein said polypeptidecomprises repeating units of at least 3 amino acids wherein saidrepeating units are connected through at least one amino acid which iscleaved by a pest gut enzyme.
 15. The transformed host, according toclaim 14, wherein said repeating units are connected through anarginine.
 16. The transformed host, according to claim 15, wherein saidpolypeptide is (DPAR)₄ (SEQ ID NO. 61).
 17. The transformed host,according to claim 1, comprising one or more D-amino acids.
 18. Thetransformed host, according to claim 1, which is an algae.
 19. Thetransformed host, according to claim 1, which is a Chlorella species.20. The transformed host, according to claim 19, wherein said Chlorellais a salt water species.
 21. The transformed host, according to claim20, wherein said chlorella is chlorella desicata.
 22. The transformedhost, according to claim 1, which is a yeast.